Vaccines are currently produced by a variety of methods. Typically, influenza vaccines are produced using fertilized chicken eggs. In the United States, the Centers for Disease Control will select three virus strains which are thought to represent the most likely viruses to strike in a particular flu season. Samples of the selected viruses are provided to manufacturers as seed virus stocks which possess the desired antigenic characteristics. The seed viruses are injected into fertilized chicken eggs. These eggs are incubated while the influenza viruses multiplies. After a suitable period of time the eggs are opened and the egg white is harvested. This sample contains the viruses. The viruses are purified from the egg material and inactivated. The individual virus stocks are then combined to create the common influenza vaccine, which is typically a trivalent vaccine.
There are a variety of problems which can occur which can compromise an entire vaccine batch. For example, problems with sterility lead to the decertification of Chiron's vaccine production facility in 2004. This situation illustrates how unreliable traditional vaccine production methods can be. Moreover, current influenza vaccine production methods employ the use of hundreds of millions of chicken eggs each year. The storage, handling, and processing steps are time consuming and labor intensive. Additionally, given the long production times, if the if a new strain of influenza virus became predominant during a flu season, current egg based production methods would take several months for a new vaccine to be produced.
In view of these limitations, a more flexible and efficient method of producing antigenic material, such as an influenza vaccine is sorely needed.
Recombinant Fungal Expression of Proteins
The cloning and expression of heterologous genes in fungi has been used to produce a variety of useful proteins. For example: Lambowitz, U.S. Pat. No. 4,486,533, discloses the autonomous replication of DNA vectors for filamentous fungi by mitochondrial plasmid DNA and the introduction and expression of heterologous genes into Neurospora; Yelton et al., U.S. Pat. No. 4,816,405, discloses tools and systems that enable the modification of important strains of filamentous ascomycetes to produce and secrete large quantities of desired heterologous proteins; Buxton et aL, U.S. Pat. No. 4,885,249, discloses the transformation of Aspergillus niger by a DNA vector that contains a selectable marker capable of being incorporated into the host A. niger cells; and McKnight et al., U.S. Pat. No. 4,935,349, discloses a method for expressing higher eukaryotic genes in Aspergillus involving promoters capable of directing the expression of a heterologous gene in Aspergillus and other filamentous fungi. Similar techniques have been used to clone the mtr gene involved with amino acid transport in Neurospora crassa (“N. crassa”) and to verify the tight linking of the cloned DNA to genomic markers flanking this gene in vivo. Stuart, W. D. et al., Genome (19&9) 30:198-203; Koo, K. and Stuart, W D. Genome (1991) 34:644-651.
Filamentous fungi possess many characteristics which make them good candidates for use in producing eukaryotic proteins. Filamentous fungi can secrete complex proteins; correctly fold three dimensional proteins including disulfide bond formation; proteolytically clip proteins following translation; and glycosylate proteins using N-linked and O-linked glycosylation reactions. These abilities have made this group of organisms attractive hosts for the production of secreted recombinant proteins. (MacKenzie, D. A. et al., J Gen Microbial (1993) 139:2295-2307; Peberdy, J. F., Trends in BioTechnology (1994) 12:50-57).
Neurospora crassa has been used as a host cell for recombinant homologous and heterologous protein production. (Carattoli, A., et al., Proc Nat Acad Sci USA, (1995) 92:6612-6616; Yamashita, R. A. et al., Fungal Genetics Newsletter (1995 Suppl.) 42A; Kato, E. et al., Fungal Genetics Newsletter (1995 Suppl.) 42A; Buczynski, S. et al. Fungal Genetics Newsletter (1995 Suppl.) 42A, Nakano, E. T. et al. Fungal Genetics Newsletter (1995 Suppl.) 40:54 0). In addition, Neurospora crassa has been used as a host cell for expressing recombinant heterodimeric and multimeric proteins by means of a heterokaryon, U.S. Pat. No. 5,643,745 July, 1997, Stuart 435/69.1.